P1379: DEVELOPMENT OF A GENE THERAPY CELL DEATH-INDUCING SYSTEM REGULATED BY MICRORNAS FOR T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA.
Authors: Mafalda Duque, Joana Ferreira Da Silva, João T. Barata, Rita Fragoso
Journal: HemaSphere (2023)

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy. Although outcome has improved throughout the years, the aggressive chemotherapeutic regimens required for treatment efficacy often lead to toxicities. Moreover, a significant fraction of T-ALL patients relapse and have extremely poor prognosis. MicroRNAs (miRs) are small non-coding RNAs (~22 nucleotides) that regulate gene expression. Importantly, specific miR expression profiles have been shown to distinguish between tumor and normal cells and tumors from different developmental origins and stages of differentiation. Aims: We aim at taking advantage of T-ALL-specific miR expression profiles to develop a gene therapy cell death-inducing system (miRTo) that uses cell-endogenous miRs to regulate the expression of a killing gene specifically in T-ALL cells (miRTo applied to T-ALL, miRToTALL). This innovative strategy has the potential to circumvent the lack of specificity in T-ALL therapy and may be applied to other cancer types. Methods: miRTo design consists of a bidirectional lentiviral (LV) vector, in which the killing gene is expressed by one promoter (EF1α), and an inhibitor of the killing gene promoter is expressed by the second promoter (PGK), resulting in the constitutive repression of the system. For the development of miRToTALL, the expression of both mRNAs will be regulated by T-ALL-specific miRs. We built a prototype of miRTo using as backbone a bidirectional LV vector and used the Lac operon system as the repressor. Briefly, we cloned Lac operator sequences (LacO) downstream of the EF1α promoter and cloned the Lac repressor (LacI) downstream of the PGK promoter. For proof-of-concept purposes, we used a reporter gene, GFP, as a surrogate marker for the killing gene. Using a provisional T-ALL-specific miR expression profile (obtained by comparing miR expression in T-ALL cells versus a limited number of normal cells), we have built trial versions of miRToTALL, using different combinations of miR target sequences (TS). We cloned in tandem 4 TS of miRs specifically downregulated in T-ALL cells (miR-29a and miR-149) downstream of GFP, and cloned 4 TS of miRs specifically upregulated in T-ALL cells downstream of LacI (miR128a and miR153). Results: Upon confirming that the LacO sequences inserted in the EF1α promoter and the LacI protein expressed by the PGK promoter are functional, we evaluated the efficiency and specificity of the different miRToTALLs to selectively target leukemia cells in vitro. We performed cultures of T-ALL and non-T-ALL cells transduced with miRToTALL and analyzed GFP expression by flow cytometry. In target T-ALL cells, miRToTALL prototypes with TS for miR-128 and/or miR-153, and in particular those without TS for miR-29a, induce GFP expression. In contrast, in non-target cells, miRToTALL prototypes with TS for miR-29a and/or miR-149, and in particular those without target sites for miR-128 did not induce GFP expression. Summary/Conclusion: Our results validate the feasibility and effectiveness of miRTo technology. We have now established T-ALL-specific miR signatures by bioinformatics analysis of publicly-available datasets obtained from T-ALL and normal cells (comprising 615 samples and 2620 miRs). These signatures are currently being validated by ddPCR. Furthermore, we have preliminary data on potential killing genes effects on T-ALL cells. Upon signature validation and killing gene selection, the miRTo technology is to be tested both in vitro and in vivo Keywords: T-ALL, Targeted therapy, Gene therapy